We seek to understand the control of protein folding. Cell stress proteins, like the chaperonin GroEL, bind and protect partly folded proteins and prevent misfolding. Controlled release then leads to native conformations. Chaperonins respond to disease, and they are used diagnostically, e.g. for breast cancer. We seek molecular details of GroEL interactions with the enzyme rhodanese-an ideal substrate whose structure and folding are well documented. What general interactions allow GroEL to bind diverse partially-folded but not native proteins? We speculate that the unusual macromolecular recognition displayed by GroEL involves induction of hydrophobic exposure by ionic interactions. We will use functional and biophysical methods to study the complexes of GroEL, its monomers and apical domains that can be formed with rhodanese and the co-chaperonin, GroES. We stress techniques that respond to dynamics and conformation changes in ways that complement static, high resolution methods. The specific Aims are designed to extent ongoing research and test four hypotheses: I. Chaperonin function involves the interplay of GroEL inter-monomer interactions, intra-monomer flexibility, ATP hydrolysis, and dynamic binding of ions, proteins and GroES. Fluorescence and tritium exchanges will monitor flexibility; and hydrodynamic methods will monitor size and quaternary structure. Exchange of GroES and ADP from complex will be compared to test for rate limiting GroEL conformational changes. II. Hydrophobic surfaces on GroEL provide the general interaction for protein binding, and they can be induced and modulated by nucleotides, ions and amphiphiles including proteins and peptides. Fluorescent probes will be used to compare hydrophobic exposure on native or mutant GroEL, its monomers and isolated apical domains. Probes will be used alone, coupled to proteins or photoincorporated into relevant sites. III. Specific interacting regions on target proteins and on GroEL can be identified. We will use cross linking and bisANS photoincorporation to identify binding sites within complexes formed between GroEL and rhodanese mutants or synthetic peptides representing stabilized helices. IV. The conformation stabilities and structures of intermediates formed by partially folded target proteins influence interactions with GroEL. We will study: a) GroEL binding of rhodanese mutants with different stabilities; and b) heterogeneity of bound intermediates.